Mature Erythroid Progenitors Research Articles

Vhl deletion in Dmp1-expressing cells alters MEP metabolism and promotes stress erythropoiesis

IntroductionErythropoiesis occurs in a specialized niche in the bone marrow and is regulated in part by oxygen-sensing signaling pathways. VHL is an E3 ubiquitin ligase that regulates the degradation of hypoxia-inducible factor (HIF) proteins under normoxic conditions. In hypoxia, VHL is inactivated resulting in enhancement of HIF signaling. Methods and resultsTo investigate the effects of HIF-activation in bone cells on erythropoiesis, we performed longitudinal analyses of conditional Vhl knockout mice (Dmp1-Cre;Vhlfl/fl; VhlcKO) mice, in which Vhl is deleted primarily in osteocytes but is retained in hematopoietic cells. VhlcKO mice display elevated red blood cell counts and erythropoietin levels starting at 10 weeks of age, resembling polycythemia. Using flow cytometry, we observed increased frequency of CD71loTER119hiFSClo orthochromatophilic erythroblasts and reticulocytes in 10- and 24-week-old VhlcKO bone marrow. Features of extramedullary hematopoiesis were observed in the spleen, including red pulp hyperplasia, the presence of megakaryocytes, and increased frequency of basophilic and polychromatophilic erythroblasts and of mature stress erythroid progenitors. To investigate the mechanisms that drive the accelerated erythropoiesis in the bone marrow, we enumerated myeloid progenitors and observed higher frequencies of megakaryocyte-erythroid progenitors (MEPs) at 6 and 10 weeks of age, but MEP frequency was similar to controls at 24 weeks. Despite this normalization in MEP frequencies, bulk RNA-Seq of MEPs at this age showed upregulation of Epas1 (Hif2a), consistent with a response to hypoxia, as well as genes involved in erythrocyte development. Surprisingly, genes involved in the response to glucose were also upregulated in VhlcKO MEPs. DiscussionOur studies implicate that HIF-driven alterations in skeletal homeostasis drive changes to the bone marrow microenvironment that influence erythroid cell development and MEP metabolism.

Erythroid Progenitor Cells in the Murine Bone Marrow: Parallels with Human Counterparts and Response to Acute Anemia

In mammals, the erythron consists of lineage-specific progenitor cells that transition to morphologically identifiable precursors that ultimately enucleate to become mature red blood cells. Early- and late-stage erythroid progenitors, BFU-E and CFU-E respectively, were originally defined by their capacity to generate colonies of erythroid cells in semisolid media. We have refined a flow cytometry-based strategy based on Pronk, et al. (2007) and Tusi, et al. (2018) that parallels similar strategies developed for human erythroid progenitor cells (Yan et al., 2021) to facilitate identification and isolation of progressive stages of primary murine erythroid progenitors, termed EP1 to EP4, in the bone marrow. Sorted erythroid progenitor (EP1 to EP4) populations were cultured in colony-forming assays. EP1 consists almost entirely of BFU-E, while EP4 was highly enriched in CFU-E, and the intermediate EP2 and EP3 populations progress from immature to more mature erythroid colony-forming progenitors. Global RNA-Seq studies were performed on three replicates of primary bone marrow EP1-4 from 9-12 week old outbred mice. EP1/2 and EP3/4 each shared the most similarities, consistent with the enrichment of BFU-E in the former and CFU-E in the latter. We also analyzed the global transcriptome of EP1-4 derived from in vitro culture of human CD34+ hematopoietic stem and progenitor cells to compare with the murine counterparts. Approximately two-thirds of differentially expressed genes displayed similar patterns as murine and human EP1 transition progressively to EP4. The largest transition in gene expression occurred between EP2 and EP3. Analysis of genes differentially expressed between EP2 and EP3 both in human and in mouse (adj. p-value <0.05, 361 down and 242 up) revealed marked downregulation of transcription factors expected from previous studies of erythroid progenitors (Gata2, Cd34), as well as those associated with multipotential, myeloid, and megakaryocyte lineages (Sp1 and Runx1). Upregulated transcription factors included prototypical factors associated with erythropoiesis (Klf1, Gata1 and Tal1), as well as Srebf1, a central regulator of cholesterol homeostasis. In both species the transition from EP2 to EP3 was characterized by downregulation of the gene ontology terms associated with myeloid and megakaryocyte development, and the upregulation of gene ontology terms associated with erythrocyte development, cell cycle, heme metabolism, and VLDL particle assembly. Interestingly, cholesterol content increases as EP1/2 transition to EP3/4 in both primary murine and human bone marrow. These data indicate a shared progression of erythroid progenitors can be proactively delineated in human and mouse systems. To better define the response of erythroid progenitors to stress, we established an acute anemia model in mice, reproducibly lowering the hematocrit by half via phlebotomy. Induction of acute anemia leads to a marked expansion of EP3 and EP4 at 48 hours both in the bone marrow and in the spleen. Additionally, these expanded EP3/4 populations were characterized by an increase percentage of the cells in S-phase. Principal component analysis of global transcriptomic studies of EP1-4 isolated from the marrow 48 hours after the induction of anemia revealed parallel differentiation programs when compared to their steady-state murine counterparts. 70% of the shared genes upregulated in the transition from EP2 to EP3 were upregulated to a greater extent in response to anemia. Upregulated genes, particularly in EP3/4, included known EPO-responsive genes (Pim1, Socs2, Tfrc, and Cited4), as well as hypoxia-associated genes. Interestingly, key cholesterol biosynthesis genes (Hmgcr, Hmgcs1, and Sqle) were also further upregulated in anemic versus steady-state EP3/4. This latter finding suggests that cholesterol homeostasis may play a key role in the erythroid progenitor response to stress. We conclude that late-stage erythroid progenitor (EP3/4) expansion is a specific and integral component of regenerative erythropoiesis. The identification and isolation of stage-specific murine erythroid progenitor cells that are highly analogous to their human counterparts will facilitate an improved understanding of normal and disordered erythropoiesis.

Transcriptional Control of Gene Expression and the Heterogeneous Cellular Identity of Erythroblastic Island Macrophages.

During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.

Comprehensive transcriptome analysis of erythroid differentiation potential of olive leaf in haematopoietic stem cells.

Anaemia is one of the leading causes of disability in young adults and is associated with increased morbidity and mortality in elderly. With a global target to reduce the disease burden of anaemia, recent researches focus on novel compounds with the ability to induce erythropoiesis and regulate iron homeostasis. We aimed to explore the biological events and potential polypharmacological effects of water‐extracted olive leaf (WOL) on human bone marrow–derived haematopoietic stem cells (hHSCs) using a comprehensive gene expression analysis. HPLC analysis identifies six bioactive polyphenols in the WOL. Treatment with WOL for 12 days regulated gene expressions related to erythroid differentiation, oxygen homeostasis, iron homeostasis, haem metabolism and Hb biosynthesis in hHSCs. Functional clustering analysis reveals several major functions of WOL such as ribosomal biogenesis and mitochondrial translation machinery, glycolytic process, ATP biosynthesis and immune response. Additionally, the colonies of both primitive and mature erythroid progenitors, CFU‐E and BFU‐E, were significantly increased in WOL‐treated hHSCs. The expressions of erythroid markers, CD47, glycophorin A (GYPA), and transferrin receptor (TFRC) and adult Hb subunits‐HBA and HBB were also confirmed in immunofluorescent staining and flow cytometer analysis in WOL‐treated hHSCs. It is well known that induction of lineage‐specific differentiation, as well as the maturation of early haematopoietic precursors into fully mature erythrocytes, involves multiple simultaneous biological events and complex signalling networks. In this regard, our genome‐wide transcriptome profiling with microarray study on WOL‐treated hHSCs provides general insights into the multitarget prophylactic and/or therapeutic potential of WOL in anaemia and other haematological disorders.

Isolation of Healthy F4/80+ Macrophages from Embryonic day E13.5 Mouse Fetal Liver Using Magnetic Nanoparticles for Single Cell Sequencing.

In vivo erythropoiesis occurs in the erythroblast island niche (EBI), comprising of a central macrophage that attaches to and aids the maturation of erythroid progenitors into mature reticulocytes. Macrophages in hematopoietic tissue such as embryonic fetal liver are heterogeneous and express the cell surface protein F4/80. Earlier methods of isolating F4/80+ macrophages from hematopoietic tissue relied on FACS sorting, but the relatively low numbers of F4/80+ cells obtained after FACS sometimes led to poor RNA quality. Additionally, since EBI macrophages are attached to erythroblasts, care must be taken to avoid contamination with bound erythroblasts. We have developed a novel method for isolating F4/80+ cells from E13.5 mouse fetal liver using magnetic nanoparticles, which can be performed on the lab bench. During cell suspension and homogenization, we also add a peptide that disrupts erythroid macrophage interactions and generates F4/80+ single cells free of erythroid contamination. Thus, our protocol generates a population enriched in F4/80+ cells that are healthy and ready for sensitive techniques such as single cell sequencing.

Enasidenib Drives Maturation of Human Erythroid Precursors Independently of IDH2

Acute Myeloid Leukemia (AML) remains one of the most difficult cancers to treat, with a 30% 2-year survival rate. High-throughput sequencing of AML patients has identified mutations, including FLT3, IDH1, and IDH2, for which targeted therapies have been developed. Enasidenib is an FDA-approved, first-in-class agent that preferentially inhibits IDH2-mutant activity and reduces levels of the oncometabolite 2-HG, inducing differentiation of IDH2-mutated blasts. Interestingly, greater than 50% of enasidenib-treated patients who had no objective clinical response still demonstrated improvement in their peripheral blood counts and reached RBC transfusion independence. The mechanism underlying this phenomenon is unknown but is of great clinical relevance given the high transfusion dependence and anemia-associated complications universally associated with AML. Thus, we sought to investigate how enasidenib drives normal hematopoiesis to improve quality of life and reduce morbidity in AML patients. In this study, we demonstrate that enasidenib enhances erythropoiesis from normal CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood (CB) and bone marrow. Enasidenib doubled the proportion and total number of mature CD71+/GPA+ erythroblasts after 8 days of culture with EPO, SCF, and IL-3. In the presence of EPO, enasidenib induced a gene signature characteristic of maturing erythrocytes, with increased expression of GATA1 (1.3 fold), EPOR (2 fold), and KLF1 (1.4 fold), and decreased PU.1 (0.5 fold) and GATA2 (0.7 fold). Enasidenib-treated progenitor cells further demonstrated increased hemoglobin production (1.9 fold) and morphologic characteristics of increased erythroid maturation. Next, we sought to determine if enasidenib augments erythroid differentiation through IDH2 and IDH2-dependent pathways. First, we found that other IDH inhibitors (AG-120, AGI-6780, and AG-881) did not increase erythropoiesis at doses ranging from 1-10μM. As expected for normal HSPCs, 2-HG was not present at detectable levels in either the DMSO or enasidenib-treated conditions, and addition of 2-HG (50, 200μM) did not affect the ability of enasidenib to increase the proportion of CD71+GPA+ cells. Because it is possible that enasidenib acts through inhibition of wild-type IDH2, we generated CRISPR-Cas9 engineered IDH2 knockout (KO) CD34+ cells and treated them with enasidenib. Similar to wildtype cells, IDH2 KO CB CD34+ cells demonstrated a 3.4-fold increase in %CD71+GPA+ erythroid cells. Thus, enasidenib augments erythropoiesis independently of both mutant and wildtype IDH2 pathways. We then investigated the progenitor population that enasidenib acts on to drive erythroid maturation. Enasidenib did not increase the number of BFU-E or CFU-E colonies or the proportion of BFU-E (IL3R-CD34+CD36-) and CFU-E (IL3R-CD34-CD36+) progenitors in colony forming or liquid culture assays, respectively, leading us to conclude that enasidenib acts on more mature erythroid progenitors. Indeed, treating sorted mature CD71+ erythroid progenitors with enasidenib increased %CD71+GPA+ cells compared to DMSO control, whereas enasidenib treatment of CD71- early erythroid progenitors showed no effect. These observations provide evidence that enasidenib acts on CD71+ erythroid progenitors to increase late-stage erythroid differentiation. Given that CD71 allows for iron uptake into erythroid progenitors, we hypothesized that enasidenib modulates the heme biosynthesis pathway. Enasidenib inhibited the ABCG2 transporter, which effluxes protoporphyrin IX (PPIX), the direct precursor to heme, from the mitochondria and cytosol. Inhibition of ABCG2 by enasidenib could lead to PPIX accumulation within the cell, driving increased heme synthesis. To investigate this hypothesis, we treated cells with 20μM Ko143, a potent ABCG2 inhibitor, and observed a similar increase in %CD71+GPA+ cells as seen with enasidenib. Measurement of PPIX autofluorescence by flow cytometry and microscopy revealed an increase of PPIX in enasidenib-treated cells by 1.2-fold. Together, our data suggests that enasidenib drives maturation of CD71+ erythroid precursors independently of wildtype or mutant IDH2. Our results position enasidenib as a promising therapy to stimulate erythropoiesis and provide the basis for a clinical trial using enasidenib to improve anemia in a wide array of clinical contexts. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.

Modeling the Behavior of Hematopoietic Compartments from Stem to Red Cells in Murine Steady State and Stress Hematopoiesis

Maintenance of the blood cell system throughout the lifetime of an organism at steady state and in response to stress requires longevity and integrity of the hematopoietic stem cell pool (HSC). Modeling of erythropoiesis has been previously proposed by solely considering late stages of erythropoiesis, but a model based on all hematopoietic stem and progenitor compartments, efficient in steady state as well as in stress hematopoiesis, has never been proposed. Here we report an erythropoiesis model based on a steady state approach, further complexified by integrating regulatory processes in order to establish a model able to recapitulate steady state and acute stress erythropoiesis. As a first step, we defined a stochastic model (without regulation) relied upon a minimum of 6 cell-amplification compartments to ensure a steady state production of erythroid cells based on: i- the number of LT-HSC, ii- the number of terminal mitosis to enable red cell production from mature erythroid progenitor, iii-the differential probability of differentiation versus self-renewal in each progenitor compartment, and the division rates from iv- LT-HSC and v-last mature erythroid progenitor compartment. Computer analysis was performed using Python language with an optimization method (CMA-ES: Covariance Matric Adaptation- Evolution Strategy). This model mimics well what is observed in vivo with the LT-HSC, ST-HSC, MPP, CMP, MEP and mature red blood cell compartments. We next assessed the effects of an acute stress targeting mature red blood cells such as phenylhydrazine treatment (PHZ) on these different progenitor compartments, from LT-HSC to mature red blood cells. PHZ was i.p administered (one dose of 60 mg/ Kg) , and mice were analyzed at days 0,1,3, 5, 10, 16, and 28 for blood parameters, for the proportion of the different bone marrow (BM) and spleen progenitor compartments as well as their percentages into cell cycle (BrdU incorporation), and apoptosis (Annexin V labelling). PHZ treatment induced a severe anemia, characterized by a strong fall of the hematocrit (30-50%) at day 3, followed by a rapid 10-day recovery. Regarding BM progenitor compartments, our results showed that, together with a stability in the number of BM cells, PHZ treatment drastically reduced at day 3-5 all progenitor compartments with a direct flush from LT-HSC (-75%) to mature cells without modifying apoptosis, proliferation or egress of progenitors from BM to compensate the loss of mature cells. In a second phase, a gradual replenishment of all progenitor compartments, from the most mature (MEP, CMP) to intermediate (MPP) and immature (ST-HSC and LT-HSC), with oscillations around the equilibrium reached at day 28, was observed. Such recovery was accompanied by a recruitment of LT and ST-HSC into cell cycle: ¼ of all LT-HSC and ST-HSC cycling at day 10 compared to 2% in steady state. The former mathematical model was then applied to PHZ treatment, but a strong discrepancy occurred in the time course of red blood cell recovery, which was 3 times longer than what was observed in vivo (30 days instead of 10 days). We therefore added to the stochastic model regulations between the different compartments based on what is observed with cytokines in vivo and the modifications of the size of the progenitor compartments. This mathematic model, now complemented by addition of these regulations, recapitulated the evolution of the different progenitor compartments observed after PHZ treatment as well as in steady state. To our knowledge, this is the first multi-step modeling of hematopoiesis able to recapitulate steady state as well as stressed hematopoiesis, by taking into account the current progenitor compartments of the BM, and able to explain at the single cell level what happens during hematopoietic differentiation process. This is also the first model demonstrating that hematopoietic regulation just like cytokines effects is necessary during stress hematopoiesis but dispensable during steady state hematopoiesis. Modeling of hematopoiesis integrating heterogeneity of the cell populations as described here will allow us to study the effects of abnormal event integration in a cell population just like the effects of the emergence of a single cancer-initiating cell. Disclosures No relevant conflicts of interest to declare.

Nrf2 Null Mice Are Deficient in CD169+ Macrophages, Associated with Impaired Erythroid Response and Delayed Recovery from Acute Blood Loss

Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.

Terminal Erythroid Maturation Is Associated with Dynamic Changes in the Abundance of Histone Marks Associated with Active Transcription Elongation and RNA Polymerase II Pausing

Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Small Molecule Screens Identify CDK8-Inhibitors As Candidate Diamond-Blackfan Anemia Drugs

A safe and efficient therapy for patients with the bone marrow failure syndrome Diamond-Blackfan Anemia (DBA) is urgently needed. To identify novel drug candidates for DBA, a small molecule screen was performed using c-Kit positive E14.5 fetal liver cells from a DBA mouse model in which the DBA phenotype is induced upon doxycycline (DOX)-inducible silencing of Rps19, the most frequently mutated gene in DBA (Jaako et al. PMID: 21989989). Test compounds were added 24 hours after cell seeding and DOX addition. After four days the number of live metabolically active cells in each well was estimated based on quantitation of intracellular ATP. Fifteen commercial annotated small-molecule libraries (3 800 molecules) and 10 500 selected compounds from a diverse compound library were screened. Between the screens we identified 20 molecules that reproducibly increased proliferation of rps19-deficient erythroid progenitors 4-8 fold in a concentration-dependent manner. Hits from annotated libraries included inhibitors of TGFb receptor, DYRK and Casein kinases. The most potent hits however were compounds in a series of thienopyridines, with an unknown target profile, but with a core structure suggesting kinase inhibition activity. Database searches revealed that the structure-activity relationships (SAR) of 12 active and 10 inactive analogues were similar to that of a series of thienopyridines previously reported as bone anabolic agents by unknown mechanism (Saito et al. PMID: 23453217). The most potent analogues described by Saito et al. as bone anabolic agents were synthesized, and the compounds rescue also RPS19-deficient erythroid cell proliferation in a potent manner (EC50= 20-50 nM). In an attempt to identify the molecular target of these compounds, six actives including thienopyridines 15k and 15w were subjected to kinase profiling against 468 kinases (DiscoverX). Three of the compounds targeted cyclin- dependent kinases CDK8 and its paralog CDK19, and in particular the most potent molecule (15w) is a highly selective CDK8 inhibitor. To confirm whether CDK8 inhibition underlies the rescue of the DBA phenotype, structurally unrelated and potent CDK8 inhibitors including CCT-251545, Senexin A, Senexin B, and Sel120-34A were also evaluated. All tested CDK8-inhibitors rescue proliferation and erythroid maturation of c-kit+ cells from the DBA mouse in a concentration-dependent manner. To further investigate the potential of CDK8 as a therapeutic target in DBA several CDK8 inhibitors were evaluated in erythroid cultures of primary DBA patient cells. CDK8 inhibitors 15w, Senexin B and Sel120-34A increase erythroid progenitor proliferation 5-10 fold of CD34+ peripheral blood cells from three DBA patients (RPS19, RPS26, RPL35a mutations), and increase the fraction of transferrin receptor (CD71) and glycophorin A positive cells in culture. Healthy CD34+ peripheral blood treated with CDK8 inhibitors show no increase in proliferation. Finally, we show that bone marrow failure and anemia in the DBA mouse model is partially rescued (RBC and Hemoglobin levels, p<0.001) after 8 daily oral doses (30mg/kg) of CDK8 inhibitor Sel120-34A. Since CDK8 is the regulatory subunit of the Mediator complex, acting as a regulator of RNA pol II transcription the therapeutic effect of CDK8 inhibitors likely involves direct changes in gene expression. RNA-Seq and gene set enrichment analysis on DBA mouse model cells treated with CDK8 inhibitor Sel120-34A shows p53 target genes are induced in RPS19-deficient cells and that CDK8 inhibitor treatment reverses this change, agreeing with previous reports that CDK8 can regulate transcription of p53 target genes. We confirm CDK8-inhibitor treatment reverses nuclear localization of p21 in RPS19-deficient cell lines. The most significant effect in gene set analysis however is the rescue of MYC-target gene expression, including increased expression of several ribosomal protein genes. In summary, selective and potent CDK8 inhibitors dramatically improve proliferation and maturation of erythroid progenitors in an animal model for DBA and in primary DBA patient cells. The therapeutic effect involves reduced expression of p53-target genes and increased expression of MYC-target genes. Given lack of adverse effects at effective doses in vivo, further development of SEL120-34A as a treatment for DBA patients is warranted. DisclosuresRzymski:Selvita S.A.: Employment, Equity Ownership. Johansson:LU Holding: Patents & Royalties. Lundbäck:LU Holding: Patents & Royalties. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

3055 - A Single Cell Haematopoietic Landscape Resolves Eight Lineage Trajectories and Defects in Kit Mutant Mice

Haematopoietic stem and progenitor cells play an essential role in maintaining the blood system. The fate decisions in these cells are carefully balanced to produce the correct proportions of mature cell types, yet this balance can be severely disrupted in haematological diseases. To capture gene expression changes across the haematopoietic landscape, we profiled over 44,000 Kit+ mouse bone marrow cells using a droplet based single-cell RNA-Sequencing method. These molecular profiles represent a reference transcriptional landscape revealing entry points to eight different blood lineages (erythroid, megakaryocytic, mast cell, basophil, eosinophil, neutrophil, monocyte and lymphoid). Contrasting single-cell gene expression profiles from a Kit mutant mouse model (W41/W41) revealed qualitative differences in the transcriptional landscape, with a distinct mast cell entry point missing from the mutant mouse bone marrow stem/progenitor cells. Mapping W41/W41 cells to their corresponding wild-type cell types showed a quantitative shift towards more mature erythroid progenitors in the Kit mutant, which we validated using FACS analysis. Differential gene expression between the clusters highlighted potential compensatory processes including downregulation of pro-apoptosis genes in W41/W41 erythroid progenitors, and upregulation of genes in the integrated stress response pathway. We are now working on analysing these data using graph abstraction, which combines clustering analysis with trajectory inference, to better understand gene expression changes throughout the complex haematopoietic differentiation hierarchy. We expect this analysis will identify genes involved in haematopoietic cell fate decisions, and highlight genes with different dynamics in wild-type versus perturbed differentiation.

MicroRNA-22 controls interferon alpha production and erythroid maturation in response to infectious stress in mice

MicroRNA-22 (miR-22) is a highly conserved microRNA that can regulate cell proliferation, oncogenesis, and cell maturation, especially during stress. In hematopoietic stem cells (HSCs), miR-22 has been reported to be involved in the regulation of key self-renewal factors, including Tet2. Recent work demonstrates that miR-22 also participates in regulation of the interferon (IFN) response, and expression profiling studies suggest that it is variably expressed at different stages in erythroid differentiation. We thus hypothesized that miR-22 regulates maturation of erythroid progenitors during stress hematopoiesis through its interaction with IFN. We compared the blood and bone marrow of wild-type (WT) and miR-22-deficient mice at baseline and upon infectious challenge with systemic lymphochoriomeningitis (LCMV) virus. miR-22-deficient mice maintained platelet counts better than WT mice during infection, but they showed significantly reduced red blood cells and hemoglobin. Analysis of bone marrow progenitors demonstrated better overall survival and improved HSC homeostasis in infected miR-22-null mice compared with WT, which was attributable to a blunted IFN response to LCMV challenge in the miR-22-null mice. We found that miR-22 was expressed exclusively in stage II erythroid precursors and downregulated upon infection in WT mice. Our results indicate that miR-22 promotes the IFN response to viral infection and that it functions at baseline as a brake to slow erythroid differentiation and maintain adequate erythroid potential. Impaired regulation of erythrogenesis in the absence of miR-22 can lead to anemia during infection.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Participation of signaling cascades in the regulation of erythropoiesis under conditions of cytostatic treatment.

We studied the role of signaling pathways in the regulation of erythropoiesis against the background of myelosuppression caused by administration of 5-fluorouracil. The important role of cyclic AMP in the maturation of erythroid progenitors after cytostatic treatment was demonstrated. The secretory activity of myelokaryocytes during the period of erythroid hemopoiesis recovery is mainly regulated via the p38 MAPK signaling pathway; non-erythropoietin factors are involved in the formation of erythropoietic activity of adherent cells of the microenvironment.

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry workup.

While multidimensional flow cytometry (MDF) has great utility in diagnostic workups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4 Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia. Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2(V617F) positive myeloproliferative neoplasms (MPN), and 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%), and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS using a lysis protocol practical for routine implementation in clinical flow cytometric workup. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically.

Unsupervised Discovery of Early Markers of Erythroid Maturation in Human Donor Marrow

Antigenic expression during erythroid maturation is not well understood. Despite the large number of surface antigens currently described for leukocytes, only a few (e.g. CD36, CD45, CD71, CD117, and CD235a) have been well characterized on erythroid cells. Here, we apply novel bioinformatic tools to select antigens with high potential for identifying successive stages of erythroid maturation in an unsupervised and unbiased manner from a high dimensional dataset. In brief, flow cytometry was performed on 3 normal donor bone marrows using Becton-Dickinson lyoplates containing a total of 275 unique antibodies and 8 gating reagents (CD15, CD19, CD34, CD38, CD45, CD71, CD117, and CD123). Each of the 275 independent data files from the same donor was aligned using the gating reagents and a weighted nearest neighbor algorithm in order to synthesize a flow data set with 283 antigens and 50,000 events. We applied a modified SPADE algorithm to generate a maturational path for cells of the erythroid lineage starting with CD34+/CD38- progenitors through the mature erythrocyte stage. A non-parametric Kruskal–Wallis test was used to identify and rank antigens that show differential expression along the erythroid maturational sequence.To discern antigens having the greatest discrimination for early vs. late erythroid maturation, we identified 15 antigens common to the top 25 most differentially expressed antigens from each donor sample. As expected, our method correctly identified the three erythroid gating reagents (CD45, CD71 and CD117) as well as previously a previously described erythroid associated antigen (CD36). A few of the identified antigens have been described are less common but also known to be differentially expressed on erythroid cells (e.g. integrin members CD29, CD44, and CD49d), however several additional novel antigens were also identified with strong differential expression including CD46, CD58, CD81, CD98, CD99, CD164, CD220 and CD321 (Figure 1). Of note, CD49d, CD98 and CD164 are of particular interest as they appear to be gradually lost during maturation from the early normoblast through the mature erythrocyte stages. This is in contrast with the other antigens that show high expression on pronormoblasts—with rapid decline during the early normoblast stage to the level of erythrocytes. We examined selected antigens from the synthetic data of all 3 samples using manual gating, and interestingly CD99 retained expression among the erythroid cells to late stage normoblasts in 2 of the 3 samples, suggesting some element of phenotypic variability among normal individuals for this antigen.Additionally, several antigens appear useful in distinguishing erythroid lineage cells from non-erythroid precursors including beta-2 microglobulin, CD50, and HLA-ABC which all showed ubiquitous expression in non-erythroid precursors with low to absent expression among cells of the erythroid lineage.In summary, using a novel unbiased method for large-scale antigen discovery, we have identified multiple novel antigens that are differentially down regulated with progressive erythroid maturation and appear useful in further delineating erythroid progenitor maturation. Additional work is underway to correlate these antigens with our morphologic understanding of erythroid maturation. Further work in characterizing these changes in myeloid stem cell disorders is on going and may be of diagnostic utility in the diagnosis of myelodysplastic syndromes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epo-induced erythroid maturation is dependent on Plcγ1 signaling.

Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Binding of erythropoietin (Epo) to its receptor (EpoR) is required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Janus kinase 2 (Jak2) tyrosine kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR-Jak2 independently of Stat5. Plcγ1-deficient pro-erythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined HSCs (Lin(-)Sca1(+)KIT(+)CD48(-)CD150(+)) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation, we assessed changes occurring at the global transcriptional and DNA methylation level after inactivation of Plcγ1. The top common downstream effector was H2afy2, which encodes for the histone variant macroH2A2 (mH2A2). Inactivation of mH2A2 expression recapitulated the effects of Plcγ1 depletion on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target mH2A2, as a 'non-canonical' Epo signaling pathway essential for erythroid differentiation.

Assessment of erythroid dysplasia by "difference from normal" in routine clinical flow cytometry work-up.

Introduction: While multidimensional flow cytometry (MDF) has great utility in diagnostic work-ups of patients with suspected myelodysplastic syndromes (MDS), only the myeloid lineage has demonstrated reproducible abnormalities from multiple laboratories. With the effects of ammonium chloride (NH4 Cl) lysis on erythroid progenitors previously described, we applied this protocol to a patient cohort with diagnosed MDS to investigate phenotypic abnormalities that indicate erythroid dysplasia. Method: Bone marrow specimens [39 MDS, 9 acute myeloid leukemia (AML), 7 JAK2V617F positive myeloproliferative neoplasms (MPN), 5 nutritional deficiencies] were processed by NH4 Cl lysis and Ficoll preparation and evaluated by MDF using a difference from normal algorithm. Results: For the MDS cohort, phenotypic abnormalities on the mature erythroid progenitors were frequent for CD71 and CD36 (36% for each antigen); abnormalities for CD235a (8%) were observed. Among immature erythroid progenitors, abnormal maturation patterns (≤5%) and increased CD105 intensity (9%) were seen. Increased frequency of CD105 bright cells was observed (18%). While antigenic abnormalities correlated between NH4 Cl lysis and Ficoll preparation, the lysis method demonstrated the most consistent quantitative antigen intensities. Mean erythroid phenotypic abnormalities and prognostic cytogenetic subgroups correlated strongly. Morphologic and erythroid phenotypic abnormalities correlated, as did increasing FCSS and number of erythroid abnormalities, albeit without further increase for AML patients. Discussion: These data expand the understanding of erythropoiesis and define immunophenotypic abnormalities that indicate dyserythropoiesis in MDS utilizing a lysis protocol practical for routine implementation in clinical flow cytometric work-up. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. © 2014 Clinical Cytometry Society.

Analysis of the interactome of ribosomal protein S19 mutants.

Diamond-Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one-fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild-type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild-type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 (http://proteomecentral.proteomexchange.org/dataset/PXD000640).